Kinetics of rat brain and liver solubilized membrane-bound catechol-O-methyltransferase

Catechol-O-methyltransferase (COMT), an enzyme involved in the metabolism o f catecholamines, is present in mammals as soluble (S-COMT) and membrane-bo und (MB-COMT) forms. The kinetic properties of rat liver and brain solubili zed MB-COMT were evaluated and compared with the ones of the respective nat ive enzymes. Treatment with Triton X-100 did not affect the affinity of S-C OMT for the substrate (adrenaline) or the activity of the enzyme. Conversel y, solubilized MB-COMT presented a lower affinity for the substrate than th e native protein, as evidenced by a significant increase in the K-m values: 9.3 (6.2, 12) vs 2.5 (0.8, 4.3) muM for the liver enzyme and 12 (11, 13) v s 1.4 (1.0, 1.9) muM for the brain enzyme. A 1.6- and 1.5-fold increase in V-max was also observed for the liver and brain solubilized enzymes, respec tively, The actual enzyme concentrations (molar equivalence, nil,,) and the ir efficiency in the O-methylation reaction (catalytic number, K-cat) were determined from Ackermann-Potter plots. Both liver and brain solubilized MB -COMT were more efficient in methylating adrenaline than the respective nat ive enzymes as revealed by higher K-cat values (P < 0.05): 16.4 +/- 0.9 vs 10.9 +/- 0.8 min(-1) (brain) and 5.9 +/- 0.3 vs 3.3 +/- 0.2 min(-1) (liver) . Subjecting liver solubilized MB-COMT to further purification increased th e K-m of the enzyme to the levels of liver S-COMT, 252 (127; 377) vs 257 (1 03; 411) <mu>M. The solubilization process significantly alters MB-COMT kin etic properties but only after partial purification does the enzyme present an affinity for the subtrate identical to S-COMT. (C) 2000 Academic Press.